功能性奈米金粒子輔助之酵素固定化技術應用於葡萄糖感測電極線上流動注射分析與廢棄竹筷的連續式固定化纖維酵素 水解反應

Functionalized nano-gold particle assisted enzyme immobilization technique for glucose sensor electrode on-line flow injection analysis and continuous immobilized cellulase hydrolysis of waste bamboo chopsticks

張國鍾(Kuo-Chung Chang)

指導教授 ： 鄭建業

中原大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6840/CYCU.2013.00159

摘要

功能性奈米金粒子輔助之酵素固定化方法，已可用於葡萄糖感測電極與固定化纖維酵素之製作，並被分別應用於葡萄糖感測電極線上流動注射分析及廢棄竹筷的連續式固定化纖維酵素水解反應。本研究中，以HB型筆蕊做為新式葡萄糖感測電極之基材，先於碳電極表面修飾上碳膏及金粒子，再將L-半胱胺酸鍵結到碳電極表面上的金粒子，最後，以化學鍵結方式將葡萄糖氧化酵素及電子媒介物接到L-半胱胺酸。此種電極具有寬廣的葡萄糖標準添加校正曲線之線性範圍(0-39.0 mM, r2=0.9956)，低偵測極限值(7.8 μM)，5秒的快速感測時間，感測靈敏度則是2212 nA mM-1，並可重複使用50天以上(重複使用的最長時間是94天)。此種酵素電極透過特別設計的流動槽結合流動注射分析系統線上監測固定化纖維酵素水解廢棄竹筷反應產生之葡萄糖產物，在反應32小時，由水解反應所產生之最大葡萄糖產量是0.46 mM。甘露糖、半乳糖、纖維雙糖及木糖對此種酵素電極感測的干擾程度分別為7.0%、5.0%、0%及0%。這種葡萄糖感測電極線上流動注射分析技術對水解反應產生之葡萄糖的量測亦具有良好的準確度(94.4-97.7%)及精確度(RSD 2.2-4.5%)。統計分析顯示葡萄糖感測電極線上流動注射分析系統與傳統的高效液相層析法分析葡萄糖並無明顯系統誤差存在。
同樣地，此種酵素固定化技術也用於固定化纖維酵素之製作。以鹽酸活化矽石，3-硫醇丙烷基三甲氧基矽烷((3-mercaptopropyl) trimethoxysilane, 3-MPTMS)及奈米金粒子再修飾於活化矽石上，然後將L-半胱胺酸鍵結於活化矽石上的奈米金粒子，最後，以化學鍵結方式將纖維酵素固定化在活化矽石表面的L-半胱胺酸，形成一種新式之固定化纖維酵素。此種固定化纖維酵素亦用於廢棄竹筷的連續式水解反應產生葡萄糖。廢棄竹筷的固定化纖維酵素水解反應最佳條件是pH 8.0及50℃。廢棄竹筷粉末的連續式水解反應是以0.3gL-1的初始廢棄竹筷粉末濃度，酵素承載量40 mg纖維酵素 (g矽石)-1的固定化纖維酵素，0.5 mL min-1的流動速率連續進樣0.2 g L-1的廢棄竹筷粉末溶液與連續抽取出水解反應液進行四天，重複連續式水解反應的纖維素之葡萄糖轉換率(72.0-76.6%)及累積的總重量(630.5-671.2 mg)的結果都比批次式(137.8 mg及45.9%)水解反應好。於較高的酵素承載量(117 mg 纖維酵素 (g 矽石)-1)的固定化纖維酵素，纖維素之葡萄糖轉換率更高達82.9%及1418 mg總累積量。另外，此種固定化纖維酵素亦可藉由過濾方式輕易地回收，在經過六個循環(90天以上)的連續式水解反應之後，其酵素活性仍然可以保持甚至超過初始的活性。

關鍵字

葡萄糖感測電極 ； 葡萄糖 ； 線上流動注射分析 ； 固定化纖維酵素 ； 連續式水解反應 ； 廢棄竹筷 ； 功能性奈米金粒子

並列摘要

Functionalized nano-gold particles assisted enzyme immobilization has been used for preparations of glucose sensor electrode and immobilized cellulase which have been applied for on-line flow injection analysis (on-line FIA) of glucose from waste bamboo chopsticks powder hydrolysis and continuous hydrolysis of waste bamboo chopsticks powder, respectively. In this study, the HB type pencil lead was used as the material for the new type glucose sensor electrode. The surface of the carbon electrode was modified with carbon paste and gold particles first. Then, L-cysteine was bound to the gold particle on the carbon electrode surface. Finally, the glucose oxidase and mediator was bound to the L-cysteine on the carbon electrode surface with chemical covalent bond. This kind of electrode possessed a wide linear range (0-39.0 mM, r2=0.9956) of glucose standard addition calibration curve, low detection limit (7.8 μM), and fast response time (5 s). The sensitivity was 2212 nA mM-1. The glucose sensor electrode can be used more than 50 days (the longest time was 94 days). This enzyme electrode was coupled to the FIA system through a specifically designed flow cell for on-line monitoring the glucose product from waste bamboo chopsticks hydrolysis by immobilized cellulase. The maximum amount of the glucose produced from the hydrolysis was 0.46 mM at 32 h reaction time. The interference level for the enzyme electrode measurement was 7%, 5%, 0% and 0% for mannose, galactose, cellobiose and xylose, respectively. The technique of the glucose sensor electrode on-line FIA also has good analytical accuracy (94.4-97.7%) and precision (RSD 2.2-4.5%) for glucose measurement. The statistical analysis shows that no obvious systematic error exists between the glucose sensor electrode on-line FIA system and traditional HPLC system for glucose analysis.
Similar enzyme immobilization method as the glucose sensor electrode was also used for the preparation of immobilized cellulase. Silica was activated with hydrochloric acid first. Then, the activated silica was reacted sequentially with (3-mercaptopropyl) trimethoxysilane (3-MPTMS) and nano-gold particles. Thus, L-cysteine can be bound to the nano-gold particle through its thio group. Finally, cellulase was immobilized on L-cysteine modified silica surface through the peptide-bond coupling reagent, N, N’-dicyclohexyl carbodiimide to form the immobilized cellulase. This immobilized cellulase was used to produce glucose by the continuous hydrolysis of waste bamboo chopsticks powder. The optimal conditions for the immobilized cellulase hydrolysis of waste bamboo chopsticks were pH 8.0 and 50C. The continuous hydrolysis of waste bamboo chopsticks powder was performed with an initial 0.3 g L-1 waste bamboo chopsticks powder, a cellulase loading of 40 mg cellulase (g silica)-1, a continuous feed concentration of 0.2 g L-1 waste bamboo chopsticks powder at a continuous feed and draw rate of 0.5 mL min-1, and a reaction period of 4 days. The conversion rates of the repeated continuous hydrolysis of waste bamboo chopsticks powder were 72.0-76.6% and the corresponding total accumulated amounts of glucose were 630.5-671.2 mg which were much better than the batch hydrolysis (45.9% and 137.8 mg). At higher enzyme loading (117 mg cellulase (g silica)-1) the conversion rate was 82.9% and a total amount of 1418 mg glucose. The immobilized cellulase can be recovered easily by filtration. For six repeated continuous hydrolysis cycles for a period of 90 days, the enzyme activity is kept about the same or even better than the initial activity.

並列關鍵字

functionalized nano-gold particle ； glucose ； continuous hydrolysis ； waste bamboo chopsticks ； on-line flow injection analysis ； immobilized cellulase ； glucose sensor electrode

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